Isoprene is an important monomer for the production of specialty elastomers including motor mounts/fittings, surgical gloves, rubber bands, golf balls and shoes. Styrene-isoprene-styrene block copolymers form a key component of hot-melt pressure-sensitive adhesive formulations and cis-poly-isoprene is utilized in the manufacture of tires (Whited et al. Industrial Biotechnology 2010 6(3):152-163). Manufacturers of rubber goods depend on either imported natural rubber from the Brazilian rubber tree or petroleum-based synthetic rubber polymers (Whited et al. 2010, supra).
Given an over-reliance on petrochemical feedstocks, biotechnology offers an alternative approach to the generation of industrially relevant products, via biocatalysis. Biotechnology offers more sustainable methods for producing industrial intermediates, in particular isoprene.
There are known metabolic pathways leading to the synthesis of isoprene in eukaryotes such as Populus alba and some prokaryotes such as Bacillis subtillis have been reported to emit isoprene (Whited et al. 2010, supra). Isoprene production in prokaryotes is however rare, and no prokaryotic Isoprene synthase (hereafter ISPS) has been described to date.
Generally, two metabolic routes have been described incorporating the molecule dimethylallyl-pyrophosphate (—PP), the precursor to isoprene. These are known as the mevalonate and the non-mevalonate pathways (Kuzuyama Biosci. Biotechnol. Biochem. 2002 66(8):1619-1627), both of which function in terpenoid synthesis in vivo. Both require the introduction of a non-native ISPS in order to divert carbon to isoprene production.
The mevalonate pathway generally occurs in higher eukaryotes and Archaea and incorporates a decarboxylase enzyme, mevalonate diphosphate decarboxylase (hereafter MDD), that introduces the first vinyl-group into the precursors leading to isoprene. The second vinyl-group is introduced by isoprene synthase in the final step in synthesizing isoprene. The non-mevalonate pathway or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway occurs in many bacteria and dimethylallyl-PP is generated alongside isopentenyl-PP, two molecules which are interconvertible via the action of isopentenyl pyrophophate isomerase or isopentyl diphosphate isomerase (hereafter IDI).